Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

Abstract

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca 2؉ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca 2؉ transients. release mediated by these two channels (for review see Refs. 1-4). These proteins, including calsequestrin (Csq), calmodulin, triadin, junctin, Junctophilins 1 and 2, MG-29, FKBP12, and others yet to be discovered, along with the RyR and DHPR make up the so-called calcium release units (CRUs) (5).Triadins, a multimember family of proteins that are the product of alternative splicing from a single gene and expressed almost exclusively in striated muscle (3, 6), have generated significant attention in recent years for their involvement in a variety of cellular events in muscle cells, but their precise role in muscle function is mostly unknown. Triadin was first identified in skeletal muscle as a 94-to 95-kDa transmembrane protein (7,8) that is abundantly expressed on the junctional sarcoplasmic reticulum (jSR), were it colocalizes with RyR1 and DHPR (9).Early studies of binding assays of solubilized SR proteins showed that triadin could not only be coimmunoprecipitated with other triadic proteins (10) but also could associate into macromolecular complexes with both the DHPR and RyR1 (7,11,12). Based in this association triadin was proposed as the key molecular linker mediating the DHPR/RyR1 communication during muscle contraction. Although functional interactions between triadin and the DHPR in skeletal muscle have proven difficult to confirm, functional and structural interactions between triadin and RyR1 have been documented by several investigators. In vitro studies have shown that the SR luminal domain of triadin not only interacts with RyR1 but appears to anchor Csq to it, mediating the functional coupling between these two proteins via specific domains (13-17).Several studies have suggested a major role for triadin 95 in modulating RyR channel properties. Both an anti-triadin anti-* This work was supported by American Heart Association Grant 0530250N (to C. F. P.) and National Institute of Health Grant PO1AR47605 (to P. D. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Together these studies suggest a negative regulatory role for triadin on RyR...

Ablation of Skeletal Muscle Triadin Impairs FKBP12/RyR1 Channel Interactions Essential for Maintaining Resting Cytoplasmic Ca2+

“…Although originally thought to be the link between RyR1 and DHPR (6 -8) and a key modulator of EC coupling (9 -11), it appears evident now that triadin acts primarily as a negative regulator of RyR1 activity (12)(13)(14)(15). Although the mechanism by which triadin regulate RyRs remains unclear, there is support for the hypothesis that triadin is involved in facilitating the cross-communication be-tween calsequestrin (CSQ) and the RyRs (16 -18).…”

Triadin and CLIMP-63 form a link between triads and microtubules in muscle cells

“…Because of its ability to shape ER and SR membrane , triadin could be involved in the overall structuration of SR cisternae at the triad. In addition, triadin knockout leads to deformations of part of the triads and to a reduction in excitation-contraction coupling and muscle strength (Oddoux et al, 2009;Shen et al, 2007). Therefore, the ER and SR membrane-shaping properties of triadin might favor the RyR1-DHPR coupling.…”

Triadin mutations - a cause of ventricular arrhythmias in children and young adults

“…Skeletal muscles from two different TRDN KO mice lines were found to show~30% of triads (structures formed by a t-tubule and adjacent terminal cisterna of the SR, either side of the t-tubule) in a longitudinal or oblique instead of transverse orientation and exhibited reduced Ca 2+ release [11,12]. Some experiments also supported the occurrence of muscle weakness (reduced force) [12].…”

“…Experiments employing TRDN 'knockout' (KO) mice have provided insight into the roles of both skeletal muscle triadin isoforms [5,11,12]. Skeletal muscles from two different TRDN KO mice lines were found to show~30% of triads (structures formed by a t-tubule and adjacent terminal cisterna of the SR, either side of the t-tubule) in a longitudinal or oblique instead of transverse orientation and exhibited reduced Ca 2+ release [11,12].…”